DNA-coated Functional Oil Droplets

Many industrial soft materials often include oil-in-water (O/W) emulsions at the core of their formulations. By using tuneable interface stabilizing agents, such emulsions can self-assemble into complex structures. DNA has been used for decades as a thermoresponsive highly specific binding agent between hard and, recently, soft colloids. Up until now, emulsion droplets functionalized with DNA had relatively low coating densities and were expensive to scale up. Here a general O/W DNA-coating method using functional non-ionic amphiphilic block copolymers, both diblock and triblock, is presented. The hydrophilic polyethylene glycol ends of the surfactants are functionalized with azides, allowing for efficient, dense and controlled coupling of dibenzocyclooctane functionalized DNA to the polymers through a strain-promoted alkyne-azide click reaction. The protocol is readily scalable due to the triblock's commercial availability. Different production methods (ultrasonication, microfluidics and membrane emulsification) are used with different oils (hexadecane and silicone oil) to produce functional droplets in various size ranges (sub-micron, $\sim 20\,\mathrm{\mu m}$ and $> 50\,\mathrm{\mu m}$), showcasing the generality of the protocol. Thermoreversible sub-micron emulsion gels, hierarchical "raspberry" droplets and controlled droplet release from a flat DNA-coated surface are demonstrated. The emulsion stability and polydispersity is evaluated using dynamic light scattering and optical microscopy. The generality and simplicity of the method opens up new applications in soft matter and biotechnological research and industrial advances.Comment: 7 pages, 2 figures, 1 tabl

From Nonspecific DNA–Protein Encounter Complexes to the Prediction of DNA–Protein Interactions

©2009 Gao, Skolnick. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.doi:10.1371/journal.pcbi.1000341DNA–protein interactions are involved in many essential biological activities. Because there is no simple mapping code between DNA base pairs and protein amino acids, the prediction of DNA–protein interactions is a challenging problem. Here, we present a novel computational approach for predicting DNA-binding protein residues and DNA–protein interaction modes without knowing its specific DNA target sequence. Given the structure of a DNA-binding protein, the method first generates an ensemble of complex structures obtained by rigid-body docking with a nonspecific canonical B-DNA. Representative models are subsequently selected through clustering and ranking by their DNA–protein interfacial energy. Analysis of these encounter complex models suggests that the recognition sites for specific DNA binding are usually favorable interaction sites for the nonspecific DNA probe and that nonspecific DNA–protein interaction modes exhibit some similarity to specific DNA–protein binding modes. Although the method requires as input the knowledge that the protein binds DNA, in benchmark tests, it achieves better performance in identifying DNA-binding sites than three previously established methods, which are based on sophisticated machine-learning techniques. We further apply our method to protein structures predicted through modeling and demonstrate that our method performs satisfactorily on protein models whose root-mean-square Ca deviation from native is up to 5 Å from their native structures. This study provides valuable structural insights into how a specific DNA-binding protein interacts with a nonspecific DNA sequence. The similarity between the specific DNA–protein interaction mode and nonspecific interaction modes may reflect an important sampling step in search of its specific DNA targets by a DNA-binding protein

DNA methylation and DNA methyltransferases

The prevailing views as to the form, function, and regulation of genomic methylation patterns have their origin many years in the past, at a time when the structure of the mammalian genome was only dimly perceived, when the number of protein-encoding mammalian genes was believed to be at least five times greater than the actual number, and when it was not understood that only ~10% of the genome is under selective pressure and likely to have biological function. We use more recent findings from genome biology and whole-genome methylation profiling to provide a reappraisal of the shape of genomic methylation patterns and the nature of the changes that they undergo during gametogenesis and early development. We observe that the sequences that undergo deep changes in methylation status during early development are largely sequences without regulatory function. We also discuss recent findings that begin to explain the remarkable fidelity of maintenance methylation. Rather than a general overview of DNA methylation in mammals (which has been the subject of many reviews), we present a new analysis of the distribution of methylated CpG dinucleotides across the multiple sequence compartments that make up the mammalian genome, and we offer an updated interpretation of the nature of the changes in methylation patterns that occur in germ cells and early embryos. We discuss the cues that might designate specific sequences for demethylation or de novo methylation during development, and we summarize recent findings on mechanisms that maintain methylation patterns in mammalian genomes. We also describe the several human disorders, each very different from the other, that are caused by mutations in DNA methyltransferase genes

Ceramic materials lead to underestimated DNA quantifications : a method for reliable measurements

In the context of investigating cell-material interactions or of material-guided generation of tissues, DNA quantification represents an elective method to precisely assess the number of cells attached or embedded within different substrates. Nonetheless, nucleic acids are known to electrostatically bind to ceramics, a class of materials commonly employed in orthopaedic implants and bone tissue engineering scaffolds. This phenomenon is expected to lead to a relevant underestimation of the DNA amount, resulting in erroneous experimental readouts. The present work aims at *lpar;i) investigating the effects of DNA-ceramic bond occurrence on DNA quantification, and (ii) developing a method to reliably extract and accurately quantify DNA in ceramic-containing specimens. A cell-free model was adopted to study DNA-ceramic binding, highlighting an evident DNA loss (up to 90%) over a wide range of DNA/ceramic ratios (w/w). A phosphate buffer-based (800 mM) enzymatic extraction protocol was developed and its efficacy in terms of reliable DNA extraction and measurement was confirmed with commonly used fluorometric assays, for various ceramic substrates. The proposed buffered DNA extraction technique was validated in a cell-based experiment showing 95% DNA retrieval in a cell seeding experiment, demonstrating a 3.5-fold increase in measured DNA amount as compared to a conventional enzymatic extraction protocol. In conclusion, the proposed phosphate buffer method consistently improves the DNA extraction process assuring unbiased analysis of samples and allowing accurate and sensitive cell number quantification on ceramic containing substrates

Force Induced DNA Melting

When pulled along the axis, double-strand DNA undergoes a large conformational change and elongates roughly twice its initial contour length at a pulling force about 70 pN. The transition to this highly overstretched form of DNA is very cooperative. Applying force perpendicular to the DNA axis (unzipping), double-strand DNA can also be separated into two single-stranded DNA which is a fundamental process in DNA replication. We study the DNA overstretching and unzipping transition using fully atomistic molecular dynamics (MD) simulations and argue that the conformational changes of double strand DNA associated with either of the above mentioned processes can be viewed as force induced DNA melting. As the force at one end of the DNA is increased the DNA start melting abruptly/smoothly after a critical force depending on the pulling direction. The critical force fm, at which DNA melts completely decreases as the temperature of the system is increased. The melting force in case of unzipping is smaller compared to the melting force when the DNA is pulled along the helical axis. In the cases of melting through unzipping, the double-strand separation has jumps which correspond to the different energy minima arising due to different base pair sequence. The fraction of Watson-Crick base pair hydrogen bond breaking as a function of force does not show smooth and continuous behavior and consists of plateaus followed by sharp jumps.Comment: 23 pages, 9 figures, accepted for publication in J. Phys.: Condens. Matte

Using eggshell membranes as a DNA source for population genetic research

In the context of population genetic research, a faster and less invasive method of DNA sampling would allow large-scale assessments of genetic diversity and genetic differentiation with the help of volunteer observers. The aim of this study was to investigate the usefulness of eggshell membranes as a DNA source for population genetic research, by addressing eggshell membrane DNA quality, degeneration and cross-contamination. To this end, a comparison was made with blood-derived DNA samples. We have demonstrated 100% successful DNA extraction from post-hatched Black-tailed Godwit (Limosa limosa) eggshell membranes as well as from blood samples. Using 11 microsatellite loci, DNA amplification success was 99.1% for eggshell membranes and 97.7% for blood samples. Genetic information within eggshell membrane DNA in comparison to blood DNA was not affected (F-ST = -0.01735, P = 0.999) by degeneration or possible cross-contamination. Furthermore, neither degeneration nor cross-contamination was apparent in total genotypic comparison of eggshell membrane DNA and blood sample DNA. Our research clearly illustrates that eggshell membranes can be used for population genetic research

Atomic force microscopy shows that vaccinia topoisomerase IB generates filaments on DNA in a cooperative fashion

Type IB DNA topoisomerases cleave and rejoin one strand of the DNA duplex, allowing for the removal of supercoils generated during replication and transcription. In addition, electron microscopy of cellular and viral TopIB–DNA complexes has suggested that the enzyme promotes long-range DNA–DNA crossovers and synapses. Here, we have used the atomic force microscope to visualize and quantify the interaction between vaccinia topoisomerase IB (vTopIB) and DNA. vTopIB was found to form filaments on nicked-circular DNA by intramolecular synapsis of two segments of a single DNA molecule. Measuring the filament length as a function of protein concentration showed that synapsis is a highly cooperative process. At high protein:DNA ratios, synapses between distinct DNA molecules were observed, which led to the formation of large vTopIB-induced DNA clusters. These clusters were observed in the presence of Mg(2+), Ca(2+) or Mn(2+), suggesting that the formation of intermolecular vTopIB-mediated DNA synapsis is favored by screening of the DNA charge